| Blattella germanica,which belongs to Blattaria and Blattulidae,is a worldwide urban pest.This kind of pest not only pollutes food,clothes,and other human daily necessities but also carries a variety of pathogens and spreads diseases.During the search for a mating partner,mature females of Blattella germanica release two types of sex pheromones:the volatile sex hormone"blatteaquinone?3,6-dioxocyclohexa-1,4-dien-1-yl?methyl 3-methylbutanoate)"and the two contact sex pheromone‘27-hydroxy-3,11-dimethylheptacosan-2-one'and‘29-oxo-3,11-dimethylnonacosan-2-one'.The volatile pheromones mainly attract the males at a long distance.In contrast,the contact pheromones are non-volatile,so the males can identify the females at close range and produce sexual behaviors by detecting contact sex pheromones,such as raising the wings 90 degrees and flapping the wings.Antennae are one of the critical chemosensory organs of insects.Many kinds of chemosensory-related genes in insect antennae are involved in many essential behaviors such as finding mates,avoiding natural enemies,locating oviposition sites,foraging,and regulating population density.Sex pheromones play a crucial role in the procedure of insect mate and reproductive isolation.Females regularly emit it,and the male antennae of conspecific detect them accurately.At the molecular level,the molecular process of insect pheromone detection can be summarized as follow:Firstly,pheromone molecules enter water-soluble sensillum lymph through the pores from the sensilla surface.And then,they bind pheromone binding proteins?PBPs?to form a complex,including pheromone molecules and PBPs.PBP then transports sex pheromone molecules to olfactory receptors on the surface of olfactory sensory neurons,and further activates pheromone receptors,and generates electrical signals that prompt insects to respond to mating searches and mating-related reactions.Studying the molecular mechanism of pheromone detection in Blattella germanica will not only help clarify the olfactory perception mechanism of Blattella germanica but also provides design ideas and theoretical basis for environmentally friendly biological control.But there have been no reports of pheromone detection mechanism in Blattella germanica.Based on the antennae and other several tissue transcriptome sequencing data of the Blattella germanica,bioinformatics technology was adopted to obtain the tissue expression profiles of 109 odorant binding proteins?OBPs?.Two OBPs with high expression in male antennae were screened out as candidate genes for PBPs.Then,protein expression and purification in vitro were used to obtain high-purity recombinant proteins of the two candidate PBPs.Furthermore,the binding ability of two OBPs to three sex pheromone components and analogs was studied by the fluorescent competitive binding experiment.The main results are as follows:1.Tissue transcriptome of the Blattella germanicaThe Illumina Hi SeqTM second-generation sequencing platform was used to build the tissue transcriptomes.The tissues included male adult antennae,female adult antennae,adult maxillary palps,adult legs,5th instar nymph antennae.And the total base number was yield about 6.6 G.The filtered reads were 22,440,484.Among them,the number of parameter comparisons was about 84.62%.The total base number of the maxillary palps was about 7.02 G.The filtered reads were 23,391,760.The identifies with the genome was approximately 83.42%.The entire base number of the nymph antennae was about 7.11 G.The filtered reads were 39,645,373.The identifies with the genome was about 83.68%.The whole base number of female antennae was about7.37 G,and the filtered reads were 49,131,552.The identifies with the genome was approximately 84.23%.The total base number of male antennae was about 7.08 G,which filtered reads were 47,223,180.The identifies with the genome was approximately 80.08%.After conducting a large number of GO functional comparisons in various tissues,the differences between nymph antennae and leg functions were significant,and adult antennae and legs were significantly different at the odorant-binding facet.It suggests that antennae participate in a precise physiological process of odor binding,and it also shows some differences between the nymph antennae and adult antennae.2.Identification and sequence analysisPBP play a crucial role in the insect sex pheromone detection.With the enormous booming transcriptome and genomic data,it provides a data basis for identifying PBP of Blattella germanica.Genomic data of Blattella germanica published by NCBI was used to compare the reported 109 OBP gene sequences with our transcriptome data.The results showed that a total of 103 OBPs were detected in at least one tested tissue.Among them,32 were highly expressed in both male and female antennae;66 were highly expressed in maxillary palps;1 was highly expressed in nymph antennae,and 4were ubiquitous expression.In addition,6 OBPs?8 OBP fragments?have been detected any expression in any tested tissue.61.46%of OBPs were highly expressed in the maxillary palps.This shows that OBP not only participates in olfactory function in the Blattella germanica,but also plays a key role in gustation.PBP is a subclass of OBP.It is accountable for binding pheromone.In Blattella germanica,the sex pheromones were released by females,males detect them by antennae.Therefore,Bger OBPs with male antennae dominant expression was selected as candidate Bger PBPs.The results showed that the RPKM of Bger OBP26 and Bger OBP40 in the male antennae,were 3,977.55 and 17.54,respectively.They were higher than the value of the nymph antennae,the maxillary palps,and legs.And the RPKM in nymph antennae were 1605.3 and 8.08,in maxillary palps were 735.96 and 0,and in the legs were 0 and0.At the same time,q PCR analysis of Bger OBP26 and Bger OBP40 in male and female antennae showed that their expression in male antennae was 1.47 and 1.97 times that of female antennae,respectively.Therefore,these two Bger OBPs were selected as PBP candidates,and subsequent functional experiments were performed.3.The in vitro study of candidate PBPs from Blattella germanicaBased on the mature proteins of OBP26 and OBP40,the E.coli expression system was adopted to obtain a large amount of OBP recombinant proteins with greater than95%purity.The positions of protein electrophoresis were 19.10 and 19.36 k Da,respectively.They were consistent with the predicted molecular weights.Then,we determined their binding ability with blatteaquinone,blatteaquinone analogs,27-hydroxy-3,11-dimethylheptacosan-2-one',and‘29-oxo-3,11-dimethylnonacosan-2-one by using the fluorescent competitive assay.The research results showed that OBP26and 40 expressed a higher binding capacity.The LC50 values were 26.78±1.92 and26.38±4.72?M,and Ki were 241.99±1.92 and 24.72±4.72?M,respectively.And their binding affinities with contact pheromone components are relatively low.Besides,this study also tested the OBP26 and OBP40 binding ability under different p H conditions.The measurement results showed that when the p H is slightly neutral7.4,both OBP26 and OBP40 show strong binding ability with blatteaquinone,which are 2.49 times and 5.84 times higher than that of p H 5.0.The results are consistent with the binding characteristics of PBPs in most insects.PBPs bind sex pheromones at neural p H and release them at acid conditions.In the fluorescence binding experiment in which two kinds of OBP are mixed at different ratios,when the molar ratio of reaches 1?10?OBP26?OBP40?,the binding ability was significantly improved than when each OBP26 or 40 alone,which is 2.57times and 2.10 times higher.Although the ratio of 1?5 showed the lowest binding affinity to blatteaquinone.There was no significant difference from the affinity with only OBP26 or 40 alone.The first case is that the OBP binary mixture has a better affinity than an OBP alone.However,the detailed interaction mechanism between the putative PBPs needs further study.In summary,this paper firstly used molecular biology,bioinformatics,and biochemistry-related analysis techniques to identify two candidate PBP genes from Blattella germanica by transcriptome database,and determines the male and female antennal expression profiles of two candidate PBPs.Further characterized their binding features with blatteaquinone,blatteaquinone analogs,and contact pheromone components.The research results provide substantial evidence for understanding the mating mechanism of Blattella germanica,and for developing environmentally friendly,new and efficient pest behavior regulation technologies. |
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